Classic haemophilia or haemophilia A is an inherited bleeding disorder. It results from a chromosome X-linked deficiency of blood coagulation Factor VIII and affects almost exclusively males with an incidence of between one and two individuals per 10,000. The X-chromosome defect is transmitted by female carriers who are not themselves haemophiliacs. The clinical manifestation of haemophilia A is an increased bleeding tendency. Before treatment with Factor VIII concentrates was introduced the mean life span for a person with severe haemophilia was less than 20 years. The use of concentrates of Factor VIII from plasma has considerably improved the situation for the haemophilia patients increasing the mean life span extensively, giving most of them the possibility to live a more or less normal life. However, there have been certain problems with the plasma-derived concentrates and their use, the most serious of which have been the transmission of viruses. So far, viruses causing AIDS, hepatitis B, and non-A non-B hepatitis have hit the population seriously. Since then different virus inactivation methods and new highly purified Factor VIII concentrates have recently been developed which established a very high safety standard also for plasma derived Factor VIII.
Factor VIII (FVIII) is known to be expressed at very low levels in mammalian cells. Also, Factor VIII is known to be an unstable protein in serum-free or protein free medium. Addition of various substances has been used to improve the stability and titers of Factor VIII.
WO 9743436 discloses the addition of inhibitors of metal dependent inhibitors and/or chymotrypsins.
WO 88/08035 and WO 87/04187 disclose the addition of phospholipids to Factor VIII culture medium. Also the co-expression of von Willebrand Factor (vWF) is described.
US 2005 0227913 A1 discloses OPLS as an inhibitor of aggregation of Factor VIII by binding to the C2-domain (2303-2332). The less aggregated Factor VIII is claimed to be less immunogenic.
K. Hansen, M. Kjalke, P. B. Rasmussen, L. Kongerslev, and M. Ezban, Cytotechnol. 24 (3), 227-234, 1997, disclose the use of Bacitracin A and phosphatidylserine to prevent degradation of Factor VIII in medium.
WO 90/02175 A1 discloses processes producing recombinant polypeptide(s) by culturing eukaryotic cells in presence of protease inhibitors to prevent degradation of polypeptide(s).
EP 1707634 A1 discloses that substantial amounts of Factor VIII is associated with the cell surface and can be removed by washing with buffers of high ionic strength.
This being, there is still a need for improved production methods so as to improve the overall yield of Factor VIII polypeptides and/or reduce production costs.